Divalent metal ion sensors and binders

ABSTRACT

Diamino polyacetate benzene compounds are used as a selective fluorescence probe, sensor, or binders for divalent metal ions. The compounds provide for uses as divalent metal ion sensors in diagnostic applications and binders for environmental and medical treatments.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 60/766,587, filed Jan. 30, 2006, entitledZINC SENSORS AND BINDERS, which is incorporated by reference.

BACKGROUND

With increased awareness for the detrimental impact of metals on humanhealth and environment, it is highly desirable to develop more sensitiveand selective probes for the detection of metal ions in biological andenvironmental samples. A variety of divalent metal ions are known to beinvolved in the structural, catalytic, and regulatory aspects of thebiological system, and some such metal ions serve as prognostics ofcertain human diseases. For example, Cu²⁺, Zn²⁺, and Fe²⁺ have beenfound to be involved in aggregating β-amyloid peptides during the onsetof the Alzheimer's disease. However, due to the lack of metal ionspecific probes, the relative contribution of one type of metal ionversus the other in causing the disease is not clearly understood. Theinability to differentiate among different types of divalent metal ionsin biological samples has been one of the major impediments in the areaof bio-analytical chemistry.

Although there has been some success in detection of biologicallysignificant metal ions by developing fluorescence probes (e.g., fura-2for Ca²⁺), most of the probes exhibit cross reactivity for other metalions. This is not surprising since both physical and electronicproperties of these metal ions are not too disparate, and they tend toexhibit comparable binding affinities with their cognate chelatingagents. Consequently, not only synthetic (organic) probes but alsoenzymatic probes exhibit cross-reactivities among metal ions. Presently,quinoline-sulfonamide containing compounds and their derivatives areregarded to be as the “gold” standards for detecting low concentrationsof Zn²⁺, albeit such compounds also exhibit selectivity for Cu²⁺. Theorigin of such selectivity appears to be encoded by facile changes inthe coordination state of Zn²⁺ versus Cu²⁺. Unexpectedly, the inventionherein describes a method for the synthesis and use of novel Zn²⁺selective fluorescent compounds that exhibit a high specificity for Zn²⁺with low reactivity to other divalent metal ions.

BRIEF SUMMARY

The present invention described herein is a method used for thedetection of metal ions in biological or environmental samples using newcompounds that fluoresce upon binding to the metal ions. The syntheticbackbone of these compounds is a diamino polyacetate benzene that ismodified using various alkyl moieties to provide metal ion specificity.The diamino polyacetate benzene compounds synthesized exhibitpreferential binding to a select metal ion and their fluorescentproperties upon binding provide for a new class of organic compoundsthat act as selective sensors for diagnostic and detection applications.One such diamino polyacetate benzene showed high specificity as a Zn²⁺selective fluorescence probe or sensor relative to other metal ions. Thediamino polyacetate benzene compounds provide for uses as zinc selectivebinders that have utility for environmental clean-up and control of zincin health and medical treatments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Fluorescence emission spectra of compound 1.

FIG. 2: Fluorescence emission spectra of 100 μM of compound 1 in thepresence of different concentrations of Zn²⁺. K_(d) value of 3.9±1.8 μM.

FIG. 3: Fluorescence emission spectra of compound 3.

FIG. 4: Fluorescence emission spectra of compound 4.

DETAILED DESCRIPTION

In the present invention novel diamino polyacetate benzene compoundswere synthesized that demonstrated high selective properties for adivalent metal ion relative to other divalent metal ions. These diaminopolyacetate benzene compounds exhibited highly desirable properties thatwere advantageous as a zinc sensor for metal ion detection in biologicaland environmental samples containing other divalent metal ions. Otherproperties exhibited of the complexed diamino polyacetate benzenecompounds that were insoluble as compared to the readily solubleuncomplexed compound provided for utility in the removal of zinc fromenvironmental and waste materials as well as potential use as atherapeutic agent for the treatment of zinc based diseases.

Cai et al. synthesized aN,N,N′,N′-tetrakis(carboxylatemethyl)-2,6-diaminocresol compound thathad properties as a divalent metal binding probe. Cai, L.; Xie, W.;Mahmoud, H.; Han, Y.; Wink, D. J.; Li, S.; O'Connor, C. J. Inorg. Chim,Acta 1997, 263, 231-245. The structure that it formed contained a fivecoordinate trigonal bipyramidal that showed binding to only Co²⁺ orCu²⁺. The ligand-metal conjugate yielded a charge-transfer band around300 nm. The structural data showed that the primary binding involvedcarboxyl and amino groups.

In our analysis, the diamino polyacetate benzene appeared to be involvedin the coordination bond with either Co²⁺ or Cu²⁺ while the latter metalexhibited a distorted configuration. To improve spectral properties andblinding characteristics for divalent metal ions of the diaminopolyacetate benzene as a modifier of coordinate geometry, compounds weresynthesized{[3-(biscarboxymethylamino)-2-methoxy-5-methylphenyl]carboxymethylamino}aceticacid (compound 1) in which the phenolic oxygen was modified. Asdescribed herein, these novel compounds provide for improved propertiesand utility as selective sensors and binding agents in a variety ofapplications.

EXAMPLE 1 Synthesis of Novel Diamino Polyacetate Alkoxy BenzeneCompounds:Potassium-2,6-diamino-(N,N,N′,N′-tetraacetate)-4-methylanisole (Compound1)

Commercially available reagents, obtained from Acros Organics andAldrich were used as received. All solvents were distilled before use.Reactions were monitored by thin-layer chromatography (TLC) andvisualization was accomplished with a UV lamp. Reaction mixtures werepurified by column chromatography, performed with the indicated solventsusing silica gel (230-400 mesh). The R_(f) values were calculated basedon the eluents used for purification. The yields reported refer tochromatographically and spectroscopically pure compounds. The purity ofthe compounds were ascertained by GC/MS analysis (HP 5890 Series II GCfitted with HP 5971 Series Mass Selective Detector). ¹H and protondecoupled ¹³C NMR spectra were recorded on a Bruker AMX 500 MHzspectrometer at ambient temperature. The fluorescence spectra wereobtained using Jobin Yvon Horiba Fluorolog-3 spectrofluorometer. TheHEPES buffer was prepared from commercially available 1M solution of theflee acid and the J, was adjusted to 7.0 by the addition of KOH pelletsin the presence of 0.135 M NaCl. The fluorescence measurements werecarried out on 3 mL samples of the sensor and 3 μL aliquots of the 200mM metal ion solution in HEPES were added to the sample to make up thedesired metal concentrations.

2,6-Diamino-4-methylanisole hydrochloride.2-Methoxy-5-methyl-1,3-dinitrobenzene (0.050 g, 0.236 mmol) wassuspended in conc. Hydrochloric acid (1.2 mL). Tin granules (0.118 g,0.995 mmol) were added slowly to the mixture with stirring at roomtemperature. After 2 hours, the solution turned white (all the tingranules were dissolved), the solution was cooled to 4° C. The productwas collected as a white precipitate and recrystallized from hotwater-concentrated hydrochloric acid. Yield=0.037 g (70%) from 0.050 g2-Methoxy-5-methyl-1,3-dinitrobenzene. White flakes; mp 226° C. (dec);¹H NMR (DMSO) δ: 6.57 (s, 2H), 3.91 (s, broad, 6H), 3.72 (s, 3H), 2.17(s, 3H).

2,6-Diamino-4-methyl anisole. 2,6-Diamino-4-methylanisole hydrochloride(0.100 g, 0.443 mmol) was suspended in 3 mL of CH₂Cl₂ under N₂. Thesolution was cooled in ice-water bath to 0° C. Concentrated ammoniumhydroxide solution (0.4 mL) was slowly added using a syringe. Themixture was stirred for 10 mins as NH₄Cl precipitated out, and washedwith water. The organic layer was dried with anhydrous sodium sulfateand the solvent was removed under reduced pressure. GC-MS analysisindicated a pure product. Yield=0.067 g (100%) from 0.100 g2,6-Diamino-4-methylanisole hydrochloride; viscous oil; ¹H NMR (CDCl₃)δ: 6.02 (s, 2H), 3.76 (s, 3H), 3.67 (s, broad, 4H), 2.16 (s, 3H). ¹³CNMR (CDCl₃) δ: 139.8, 134.8, 132.9, 107.3, 58.7, 21.3; EI-MS, m/z (rel.intensity) 153 (M+1, 5.0), 152 (M+, 49.8), 138 (7.6), 137 (100), 124(1.7), 110 (8.8), 109 (16.4), 92 (4.3), 80 (3.2), 79 (1.2), 65 (2.7), 54(0.5).

Ethyl-2,6-diamino-(N,N,N′,N′-tetraacetate)-4-methylanisole (1′).2,6-Diamino-4-methylanisole (0.100 g, 0.799 mmol), KI (0.436 g, 2.63mmol), K₂HPO₄ (0.458 g, 2.63 mmol), and ethyl bromoacetate (0.33 mL, 2.9mmol) were mixed in a 250-mL flask with 10 mL of acetonitrile. Themixture was refluxed for 15 h under N₂ then freshly dried molecularsieves and more base were added. The mixture was refluxed for another 18h. The mixture was cooled and the solvent was removed under reducedpressure. The residue was dissolved in hexane-ethyl acetate mixture(7:3) and filtered through silica gel. The filtrate was distilled invacuo, and the residue was purified on a column of silica gel usinghexane-ethyl acetate (9:1). The fractions were distilled and the oilyproduct was crystallized from hexane-ethyl acetate (95:5) mixture togive pure products (analyzed by GC-MS). Yield=0.170 g (52%) from 0.100 gof 2,6-Diamino-4-methylanisole. White needles; mp 67-69° C.; ¹H NMR(CDCl₃) δ: 6.27 (s, 2H), 4.18 (q, J=7.2 Hz, 8H), 4.13 (s, 8H), 3.68 (s,3H), 2.19 (s, 3H), 1.27 (t, J=7.2 Hz, 12H). ¹³C NMR (CDCl₃) δ: 171.6,143.7, 141.3, 133.4, 113.5, 60.8, 59.7, 54.0, 21.9, 14.5; EI-MS, m/z(rel. intensity) 498 (M+2, 0.6), 497 (M+2, 2.6), 496 (M+, 11.2), 465(2.0), 424 (19.3), 423 (75.4), 335 (16.6), 321 (4.4), 307 (2.1), 2 93(2.4), 277 (4.0), 264 (18.1), 263 (100), 249 (3.6), 247 (3.8), 235(10.1), 219 (4.4), 191 (16.1), 175 (36.8), 162 (15.4), 161 (15.4), 148(11.2), 134 (5.8), 118 (5.3), 91 (3.5), 59 (9.8).

Potassium-2,6-diamino-(N,N,N′,N′-tetraacetate)-4-methylanisole (1).Compound 1′ (0.038 g, 0.077 mmol) was dissolved in 2 mL of MeOH understirring. Aqueous KOH (0.1 mL, 3M) was added to the mixture and refluxedfor 4 h. The reaction mixture was then cooled and the solvent removed toobtain a brown hygroscopic solid. Yield=0.040 g (96%) from 0.038 g ofcompound 1. ¹H NMR (D₂O) δ: 6.13 (s, 2H), 3.85 (s, 8H), 3.57 (s, 3H),2.16 (s, 3H). ¹³C NMR (D₂O) δ: 180.5, 145.1, 138.3, 133.6, 109.7, 59.7,57.1, 21.1.

Zinc-2,6-diamino-(N,N,N′,N′-tetraacetate)-4-methylanisole. The sodiumsalt equivalent of 1 (30 mg, 0.06 mmol) was dissolved in 0.75 mL D₂O andthe ¹H NMR was taken. Then ZnCl₂ (8 mg, 0.06 mmol) was added and thesolution was stirred. Some of the complex precipitated out and it wasfiltered before the ¹H NMR was taken again. ¹H NMR (D₂O) δ: 6.18 (s,2H), 3.89 (s, 8H), 3.63 (s, 3H), 2.22 (s, 3H).

Derivatives of compound 1 involve the phenolic oxygen by substitutingthe methyl group with groups that are alkylated, which but are notlimited to, ethyl, isopropyl, t-butyl, benzyl, substituted benzyl, andphenethyl, and electron withdrawing groups which include, but are notlimited to, trifluoromethyl and acyl(COCH₃). The acyl derivativesprovide additional oxygen that interact with the metal ion.

EXAMPLE 2 Fluorescence Profile ofPotassium-2,6-diamino-(N,N,N′,N′-tetraacetate)-4-methylanisole (Compound1)

The influence of divalent metal ions on the fluorescence spectralprofile of compound 1 is shown in FIG. 1, where the fluorescenceemission spectra of 200 μM compound 1 (λ_(ex)=300 nm) withstoichiometric concentrations of selected divalent metal ions. Compound1 has a weak fluorescence emission peak around 386-390 nm, which isdifferently affected by different metal ions. The emission intensity ofcompound 1 (at 390 nm) was barely affected in the presence of Mg²⁺ andonly slightly increased in the presence of Ca²⁺. In contrast, thefluorescence intensity of compound 1 decreases in the presence of Cu²⁺,Ni²⁺, and Co²⁺. The most dramatic effect of fluorescence profile ofcompound 1 was observed in the presence of Zn²⁺.

Surprisingly, in the presence of stoichiometric concentration of Zn²⁺the fluorescence emission intensity of compound 1 was increased by about10 fold. Such an enhancement in fluorescence intensity is notkinetically controlled as the time dependent incubation of Zn²⁺ withcompound 1 and its emission intensity was not altered. This deductionwas equally valid for the interaction of other metal ions with compound1, irrespective of their spectral modulating features.

Unexpectedly, the Zn²⁺-induced fluorescence enhancement of compound 1was maintained even in the presence of a 100 fold excess of Ca²⁺. Theunique properties of compound 1 can be utilized to detect Zn²⁺ in thephysiological milieu containing the high concentrations of Ca²⁺. Theseresults demonstrated that compound 1 was a Zn²⁺ specific fluorescentprobe that has application as a novel divalent zinc sensor.

EXAMPLE 3 Binding Kinetics ofPotassium-2,6-diamino-(N,N,N′,N′-tetraacetate)-4-methylanisole (Compound1)

To determine the magnitude of Zn²⁺ induced fluorescence spectral changesof compound 1 as well as its binding affinity, a detailedspectrofluorometric titration study was performed. In FIG. 2 (rightpanel) shows the fluorescence emission spectra of compound 1 (correctedfor the buffer) as a function of increasing concentrations of ZnCl₂. Thefluorescence emission intensity at 386 nm (λ_(ex)=300 nm) showed asaturating profile as a function of ZnCl₂ (FIG. 2, left panel). Sincethe concentration of compound 1 was comparable to the initialconcentrations of Zn²⁺, the binding constant of compound 1-Zn²⁺ complexwas calculated by a complete solution of the quadratic equation,describing their interaction. The solid line was the best fit of theexperimental data for the K_(d) value of 6.1±2.5 μM and thestoichiometry of 1:1 (i.e. 1 mole of bound Zn²⁺ per mol of compound 1).

The basis of compound 1 (vis a vis analogous compounds reported in theliterature) functioning as Zn²⁺ selective fluorescent sensor wasunexpected. It has been well established that unlike Cu²⁺, Co²⁺, andNi²⁺, which predominate either as the square planer or tetrahedralcoordination state, Zn²⁺ preferentially exists in the octahedral state.Because of its octahedral state, zinc can interact with all six groupsthat are contributed by the two iminodiacetate moieties of compound 1.The stoichiometry of compound 1-Zn²⁺ complex is equal to 1:1 (FIG. 2).

EXAMPLE 4 Solubility ofPotassium-2,6-diamino-(N,N,N′,N′-tetraacetate)-4-methylanisole (Compound1)

In contrast to readily soluble compound 1, the compound 1-Zn complex haslow solubility in water and methanol. All attempts at recrystallizationof the complex resulted in the formation of a white powder. Saturatedsolution of the complex in D₂O was subjected to 1H NMR analysis in anattempt to elucidate the structure of the complex. Comparison of the ¹HNMR of the compound 1 and its 1:1 mixture with Zn(II) indicated that thezinc binding induces a deshielding effect on all the protons,accompanied by substantial peak broadening. The broadening of the peaksimply that the complex was fluxional, which explained the poorrecrystallization properties.

Compound 1 was found to have a high solubility in the aqueous medium,which made it an ideal compound as a selective fluorescence probe(sensor) for detection of Zn²⁺ in biological samples. Moreover, its highsolubility made the compound an attractive therapeutic for the treatmentof medical diseases where an excess of Zn or Zn containing proteins is acausative agent of the disease. In contrast, the low solubility of thecomplex would be useful in extracting Zn²⁺ ions from environmentalmatrix, when high concentrations of Zn are present.

EXAMPLE 5 Synthesis of Novel Diamino Polyacetate Benzene Compounds:Sodium-1,3-diamino-(N,N,N′,N′-tetraacetate)benzene (Compound 3)

In order to determine the structural requirements for these sensors todetect Zn²⁺, compound 3 was synthesized such that only two IDA groupswere present (structurally similar to compound 1 except that the methoxygroup was absent).

Ethyl-1,3-diamino-(N,N,N′,N′-tetraacetate)benzene (3′). 0.211 g (2.00mmol) of 1,3-phenylenediamine, 1.661 g (10.0 mmol) of KI, 1.742 g (10.0mmol) of K₂HPO₄, 1.13 mL (10.0 mmol) of ethyl bromoacetate, 15 mL ofMeCN and freshly dried molecular sieves were used. The reaction wasrefluxed for 27 h. The mixture was cooled and the solvent was removedunder reduced pressure. The residue was dissolved in hexane/ethylacetate (7:3) and filtered through silica gel. The filtrate wasdistilled in vacuo and purified on column chromatography usinghexane/ethyl acetate (95:1) mixture. Yield=0.488 g (56%). Colorless oil;¹H NMR (CDCl₃) δ: 7.06 (t, J=7.9 Hz, 1H, Ar—H), 6.08 (d, J=7.9 Hz, 2H,Ar—H), 5.85 (s, 1H, Ar—H), 4.21 (q, J=7.3 Hz, 8H, C—CH₂—C), 4.10 (s, 8H,N—CH₂—C), 1.27 (t, J=7.3 Hz, 12H, —CH₃). ¹³C NMR (CDCl₃) δ: 171.2,149.3, 130.2, 103.6, 97.7, 61.3, 53.9, 14.4.

Sodium 1,3-diamino-(N,N,N′,N′-tetraacetate)benzene (3). Compound 3′(0.047 g, 0.104 mmols) was dissolved in 30 mL of MeOH/H₂O (2:1). 0.139 g(3.48 mmols) of NaOH was added. The solution was stirred at roomtemperature overnight. Evaporation of the solvent mixture was followedby suspension of the residue on methanol. The solution was filtered andmethanol was evaporated under reduced pressure. The residue wasdissolved in minimum water and dried under vacuum to remove trappedmethanol. Brown powder. Yield=0.038 g (84%). ¹H NMR (D₂O) δ: 7.04 (t,J=8.2 Hz, 1H, Ar—H), 5.88 (d, J=8.2 Hz, 2H, Ar—H), 5.59 (s, 1H, Ar—H),3.84 (s, 8H, —CH₂—). 6.98-6.96 (m, 1H, Ar—H), 6.89-6.87 (m, 2H, Ar—H),6.74-6.72 (m, 1H, Ar—H), 3.78 (s, 4H, —CH₂—), 3.77 (s, 3H, O—CH₃). ¹³CNMR (D₂O) δ: 180.2, 150.1, 130.3, 101.0, 95.0, 55.8.

EXAMPLE 6 Fluorescence Profile of Sodium1,3-diamino-(N,N,N′,N′-tetraacetate)benzene (Compound 3)

The influence of divalent metal ions on the fluorescence spectralprofile of compound 3 is shown in FIG. 3, where the fluorescenceemission spectra of 200 μM compound 1 (λ_(ex)=315 nm) withstoichiometric concentrations of selected divalent metal ions. Compound3 has a weak fluorescence emission peak around 355-360 nm, which isdifferently affected by different metal ions. The emission intensity ofcompound 3 (at 357 nm) was slightly enhanced in the presence of Mg²⁺,Ca²⁺, Cd²⁺, and Hg²⁺. In contrast, as expected, the fluorescenceintensity of compound 3 decreases in the presence of Cu²⁺, Ni²⁺, andCo²⁺. However, in the presence of Zn²⁺, the fluorescence of the compoundis enhanced significantly (similar to compound 1). Titration of compound3 with Zn²⁺ indicates a K_(d) value of 2 mM (approx.). These resultsindicate that the four acyl groups are crucial for the sensor to detectZn²⁺, while the methoxy group helps in red shift of the fluorescenceemission wavelength and a higher binding constant.

EXAMPLE 7 Derivatives that can Fluoresce Visible Light: Sodium2,6-diamino-(N,N,N′, N′-tetraacetate)-3-nitroanisole

An alkylated aminoanisole compound containing a nitro group in thearomatic ring (compound 4) was synthesized. The presence of the nitrogroup facilitates the molecular absorption in the visible region.Excitation wavelengths of 400 nm or above leading to fluorescenceemission of about 450-500 nm prevents cell damage, reduces cost ofequipment, and permits the visualization of the Zn-bound complex.

2,6-Dinitroanisole. 0.200 g (1.00 mmol) of commercially available2-chloro-1,3-dinitrobenzene was dissolved in a warm solution of 15 mlmethanol and 0.223 g (4.13 mmol) of NaOMe. The reaction was stirredovernight with continued warming. The residue was dissolved in waterafter evaporation of the solvent. The aqueous solution was extractedwith dichloromethane, dried and evaporated under reduced pressure togive 0.187 g of 2,6-dinitroanisole (95%) as yellow flakes. ¹H NMR(CDCl₃) δ 8.06 (d, J=8.2 Hz, 2H, Ar—H), 7.38 (t, J=8.2 Hz, 1H, Ar—H),4.09 (s, 3H, O—CH₃). ¹³C NMR (CDCl₃) δ 148.0, 129.4, 124.2, 65.1.

2,6-Diaminoanisole. 0.500 g (2.52 mmol) of compound 2,6-dinitroanisolewas suspended in 40 mL of water together with 2.310 g (35.3 mmol) of Znand 0.530 g (10.1 mmol) of NH₄Cl. The solution was boiled for 2.5 h.After cooling to room temperature, the solution was extracted with ethylacetate. The organic solution was dried with anhydrous Na₂SO₄ anddistilled in vacuo to obtain 0.330 g (95%) of clean 2,6-diaminoanisoleas a red oil. ¹H NMR (CDCl₃) δ 6.73 (t, J=7.9 Hz, 1H, Ar—H), 6.19 (d,J=7.9 Hz, 2H, Ar—H), 3.78 (s, 3H, O—CH₃), 3.73 (br. s, 4H, Ar—NH₂). ¹³CNMR (CDCl₃) δ 140.3, 134.9, 125.2, 106.6, 58.6.

Ethyl-2,6-diamino-(N,N,N′,N′-tetraacetate)anisole. 0.116 g (0.840 mmol)of 2,6-diaminoanisole was added to 14 mL of dried MeCN together with0.830 g (5.04 mmol) of KI, 0.881 g (5.06 mmol) of K₂HPO₄, and 571 μL(5.00 mmol) of ethyl bromoacetate. Some molecular sieves were added andthe solution was refluxed under air-free conditions (drying tube) for 33h. The resulting mixture was filtered through a Buchner funnel loadedwith SiO₂ using hexane-ethyl acetate (7:3). The filtrate was distilledand chromatographed over SiO₂ using hexane-ethyl acetate (95:5) mixture.0.330 g (81%) of ethyl-2,6-diamino-(N,N,N′,N′-tetraacetate)anisole wasobtained as white crystals. ¹H NMR (CDCl₃) δ 6.84 (t, J=8.2 Hz, 1H,Ar—H), 6.46 (d, J=8.2 Hz, 2H, Ar—H), 4.19 (q, J=7.2 Hz, 8H, C—CH₂—C),4.16 (s, 8H, N—CH₂—C), 3.70 (s, 3H, O—CH₃), 1.27 (t, J=7.2 Hz, 12H,C—CH₃). ¹³C NMR (CDCl₃) δ 171.6, 144.2, 129.4, 124.2, 124.1, 112.7,60.8, 54.0, 14.4.

Ethyl-2,6-diamino-(N,N,N′,N′-tetraacetate)-3-nitroanisole. 0.080 g(0.166 mmol) of ethyl-2,6-diamino-(N,N,N′,N′-tetraacetate)anisole wasdissolved in 2 mL of Ac₂O. The solution was cooled to 0° C. in anice-salt bath, followed by the addition of 13 μL (0.246 mmol) of HNO₃.The solution was stirred at room temperature for 1.5 h. The resultingmixture was cooled in ice, neutralized to pH 7 and warmed up to roomtemperature. The aqueous solution was extracted with dichloromethane andchromatographed over silica gel using hexane-ethyl acetate (9:1) toobtain 0.073 g (83%) ofethyl-2,6-diamino-(N,N,N′,N′-tetraacetate)-4-nitroanisole asyellow-brown paste. ¹H NMR (CDCl₃) δ 7.57 (d, J=9.3 Hz, 1H, Ar—H), 6.53(d, J=9.3 Hz, 1H, Ar—H), 4.22-4.4.18 (m, 8H, C—CH₂—C; N—CH₂—C),4.17-4.12 (m, 4H, C—CH₂—C), 4.00 (s, 4H, N—CH₂—C), 3.78 (s, 3H, O—CH₃),1.30-1.22 (m, 12H, C—CH₃). ¹³C NMR (CDCl₃) δ 170.4, 170.3, 148.8, 146.6,140.3, 139.0, 122.7, 112.6, 61.3, 61.0, 60.6, 55.1, 53.7, 14.4, 14.3.

Sodium 2,6-diamino-(N,N,N′,N′-tetraacetate)-3-nitroanisole. 0.073 g(0.140 mmols) ofethyl-2,6-diamino-(N,N,N′,N′-tetraacetate)-3-nitroanisole was dissolvedin 6 mL methanol. Sodium carbonate (0.118 g, 1.11 mmol dissolved in 5 mLwater) was added to the solution and the reaction mixture was stirredfor 1 h. The solvents were removed under reduced pressure. The residuewas dissolved in methanol and the insoluble excess sodium carbonate wasremoved by filtration. The methanol was removed under reduced pressure.Any remaining methanol was azeotroped with water. Yield 0.064 g (91%) ofSodium 2,6-diamino-(N,N,N′,N′-tetraacetate)-3-nitroanisole as brownpowder. ¹H NMR (D₂O) δ 7.67 (d, J=9.6 Hz, 1H, Ar—H), 6.32 (d, J=9.6 Hz,1H, Ar—H), 3.95 (s, 4H, —CH₂—), 3.84 (s, 4H, —CH₂—), 3.58 (s, 3H,O—CH₃). ¹³C NMR (D₂O) δ 178.3, 178.2, 151.1, 145.6, 125.9, 123.0, 112.3,104.5, 63.5, 63.3, 54.6.

EXAMPLE 8 Fluorescence Profile of Sodium2,6-diamino-(N,N,N′,N′-tetraacetate)-3-nitroanisole (Compound 4)

The influence of divalent metal ions on the fluorescence spectralprofile of compound is shown in FIG. 4, where the fluorescence emissionspectra of 200 μM compound 1 (λ_(ex)=365 nm) with stoichiometricconcentrations of selected divalent metal ions. Compound 4 has a UVabsorption maximum at 425 nm. However, excitation at this wavelengthdoes not lead to a fluorescence emission. Excitation at shorterwavelengths e.g. at 365 nm leads to a weak emission peak around 520 nm,which is differently affected by different metal ions. The emissionintensity of compound 4 (at 520 nm) has no discernable effect in thepresence of Hg²⁺, it was slightly quenched by Mg²⁺, Ca²⁺. In contrast,as expected, the fluorescence intensity of compound 4 was significantlyquenched in the presence of Cu²⁺, Ni²⁺, and Co²⁺. In the presence ofZn²⁺ and Cd²⁺, the fluorescence of the compound is substantiallyenhanced.

The description of the specific embodiments of the invention ispresented for the purpose of illustration. It is not intended to beexhaustive nor to limit the scope of the invention to the specific formsdescribed herein. Although the invention has been described withreference to several embodiments, it will be understood by one ofordinary skill in the art that various modifications can be made withoutdeparting from the spirit and the scope of the invention, as set forthin the claims. All patents, patent applications and publicationsreferenced herein are hereby incorporated by reference. Otherembodiments are within the claims.

1. A method for the detection of a metal ion in a sample using a diaminopolyacetate benzene compound as a metal ion sensor, comprising the stepsof: a. incubating a sample containing the metal ion for a sufficienttime to complex the metal ion with a diamino polyacetate benzenecompound; b. exciting the complex with a wavelength capable of causingemission of a fluorescent signal by the diamino polyacetate benzenecompound/metal ion complex; and c. detecting the fluorescent signalemitted by the complex.
 2. The method of claim 1, wherein the diaminopolyacetate benzene compound includes an attached alkoxy group.
 3. Themethod of claim 2, wherein the alkoxy group is a methoxy group.
 4. Themethod of claim 3, wherein the diamino polyacetate benzene compound iscompound
 1. 5. The method of claim 2, wherein the diamino polyacetatebenzene compound includes an attached nitro group.
 6. The method ofclaim 5, wherein the nitro group is in a meta position with respect tothe alkoxy group.
 7. The method of claim 6, wherein the diaminopolyacetate benzene compound is compound
 4. 8. The method of claim 1,wherein the diamino polyacetate benzene compound is a diaminotetraacetate benzene compound.
 9. The method of claim 8, wherein thediamino tetraacetate benzene compound is compound
 3. 10. The method ofclaim 1, wherein the metal ion is a divalent metal ion.
 11. The methodof claim 5, wherein the metal ion is selected from the group consistingof Zn²⁺, Cu²⁺, Co²⁺, Mg²⁺, Fe²⁺, and Ni²⁺.
 12. A sensor used for thedetection of a metal ion comprising a diamino polyacetate benzenecompound that emits a fluorescent signal after binding to the metal ion.13. The sensor of claim 12, wherein the diamino polyacetate benzenecompound includes an attached alkoxy group.
 14. The sensor of claim 13,wherein the diamino polyacetate benzene compound includes an attachednitro group.
 15. The sensor of claim 12, wherein the diamino polyacetatebenzene compound is a diamino tetraacetate benzene compound.
 16. Thesensor of claim 12, wherein the diamino polyacetate benzene compound iscovalently linked to a surface.
 17. The sensor of claim 16, wherein thesurface is a nanoparticle.
 18. A composition for removal of a metal ionfrom an environmental or biological fluid comprising a diaminopolyacetate benzene compound that binds to the metal ion so that thebound metal ion and compound can be removed from the fluid.
 19. Thecomposition of claim 18, wherein the diamino polyacetate benzenecompound includes an attached alkoxy group.
 20. The composition of claim19, wherein the diamino polyacetate benzene compound includes anattached nitro group.